The non-catalytic function of PARP2 in DNA repair and cancer therapy

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT PARP1 and PARP2 are DNA damage-induced poly-ADP-ribose (PAR) transferases, which are recruited to and activated by DNA breaks. Active PARP1&2 PARylate themselves and histones to promote DNA repair and chromatin relaxation. Dual-specificity inhibitors (PARPi) for PARP1 and PARP2 are currently used for the treatment of BRCA1/2-deficient breast, ovarian, pancreatic, and prostate cancers. However, severe anemia occurs in ~ one-third of patients, leading to dose reduction and premature termination of PAPRi therapy. PARPi also cause significant clonal hematopoiesis, a condition that increases the risk for myeloid dysplasia and acute- myeloid leukemia (MDS/AML). These hematopoietic toxicities are unexpected since the complete loss of Parp1 that is responsible for >80% of DNA damage-induced PARylation in cells, has no impact on hematopoiesis. To understand this potential on-target toxicity of PARPi, we generated mouse models with knockin catalytically inactive mutations in Parp2 – Poly-ADP-Ribosylation (PARylation) deficient (E534A, Parp2EA) or Mono-ADP- Ribosylation (MARylation) deficient (H404A, Parp2HA). In contrast to the normal development of Parp2-/- mice, mice expressing PARylation deficient PARP2 (Parp2EA/EA) died at embryonic day 16.5 (E16.5) with severe anemia and stage-specific blocks in erythropoiesis. The Parp2HA/HA mice are viable but display splenomegaly and chronic anemia. Meanwhile, our cell biology analyses suggest that clinically used PARPi effectively stalls PARP2, but not PARP1, on laser-induced DNA damage sites. Based on these and other observations, we hypothesize that PARP2 has PARylation-dependent structural functions that preferentially block nick ligation during DNA replication, underlie the PARP inhibitors-induced erythropoiesis defects and anemia. Specifically, we propose that catalytically inactive PARP2 were trapped on DNA breaks, especially 5’pho-nicks, where they prevent other repair factors (e.g., Ligase1) from accessing the DNA nicks. Correspondingly, like the Parp2EA/EA mice, Lig1-/- mice also succumbed to lethal anemia at E16.5. To test our hypothesis, we will Aim 1) investigate the mechanism that regulates PARP2 recruitment and dynamics at DNA damage sites using live-cell imaging and bulk biochemical assays; Aim 2) determine the impacts of catalytically inactive PARP2 on the recruitment and function of other DNA repair factors, and DNA replication in vivo and in vitro; Aim 3) characterize the mouse models expressing catalytically inactive - Parp2 and investigate how the loss of Trp53, CHK2, two genes associated clonal hematopoiesis, modulates the anemia in Parp2EA/EA models. The completion of the proposal will characterize the previously unrecognized structural functions of PARP2, address how catalytically inactive PARP2 compromises DNA replication and selectively abrogates erythropoiesis, providing the strategy to mitigate this on-targeted toxicity of PARP inhibitors by reducing PARP2 stalling.